Project Summary Central nervous system (CNS) tumors are the leading cause of death in pediatric oncology. While there have been great advancements in survival for some tumors, the presence of BRAF-mutations is correlated with a poor prognosis. Additionally, low-grade gliomas (LGG) that present in the midline result in increased neurologic impairment. The significant morbidity and mortality of these tumors highlights that new treatment strategies are urgently needed for this population. My research combines two potential strategies to improve survival in this high-risk patient population by 1) using a precision medicine approach and small molecule inhibitors targeting a pathway upregulated by specific mutation, and 2) a broader approach by inhibiting survival pathways such as autophagy. My published and preliminary data suggest autophagy inhibition in mitogen-activated protein kinases (MAPK) pathway driven CNS tumors is an effective therapeutic target across tumor types, is synergistic with MAPK pathway targeted chemotherapeutics, and can be optimized for rapid translation to clinical trials. It is vital to better understand how and why autophagy inhibition is effective in these tumors, how best to inhibit the pathway, and what additional biomarkers might be available for autophagy dependence to plan effective future autophagy inhibition trials and improve the survival of these patients. These studies will develop the pre-clinical data needed to establish and optimize autophagy inhibition as a clinical target for autophagy addicted brain tumors. Aim 1 will determine the optimal target for autophagy inhibition in BRAF mutated CNS tumors. The multi- step nature of autophagy lends itself to the several opportunities to disrupt the process. This aim will identify the most appropriate pharmacologic autophagy inhibitors to pursue for CNS tumors. Aim 2 will determine if the BRAFV600E mutation is required for autophagy dependence in CNS tumor cells, or if any cause of a dysregulated MAPK pathway is sufficient to identify autophagy dependence. This aim will provide biomarkers of autophagy dependence beyond BRAFV600E, which we have shown as a sensitive predictor of autophagy addiction and response to autophagy targeted inhibitors. It will expand the pool of patients that may benefit from clinical autophagy inhibition. Aim 3 will determine the mechanism by which autophagy inhibition overcomes BRAF inhibitor resistance mechanisms in CNS tumors. This effect is consistent across multiple tumor types and molecularly-distinct resistance mechanisms and preliminary evidence suggests this is related to regulation of key BH3 proteins and apoptosis. Elucidating the mechanism by which autophagy reverses resistance to RAF pathway inhibition will provide a better understanding of how to ensure continued response to these therapies and suggest additional combination therapies, such as BH3 mimetics, that can be successful in these patients.